Sphingosine 1-Phosphate and Isoform-specific Activation of Phosphoinositide 3-Kinase β

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elicits diverse biological responses, including angiogenesis, via the activation of G protein-coupled EDG receptors. S1P activates the endothelial isoform of nitric-oxide synthase (eNOS), associated with eNOS phosphorylation at Ser-1179, a site phosphorylated by protein kinase Akt. We explored the proximal signaling pathways that mediate Akt activation and eNOS regulation by S1P/EDG receptors. Akt is regulated by the lipid kinase phosphoinositide 3-kinase (PI3-K). We found that bovine aortic endothelial cells (BAEC) express both α and β isoforms of PI3-K, while lacking the γ isoform. S1P treatment led to the rapid and isoform-specific activation of PI3-Kβ in BAEC. PI3-Kβ can be regulated by G protein βγ subunits (Gβγ). The overexpression of a peptide inhibitor of Gβγ attenuated S1P-induced eNOS enzyme activation, as well as S1P-induced phosphorylation of eNOS and Akt. In contrast, bradykinin, a classical eNOS agonist, neither activated any PI3-K isoform nor induced eNOS phosphorylation at Ser-1179, despite activating eNOS in BAEC. Vascular endothelial growth factor activated both PI3-Kα and PI3-Kβ via tyrosine kinase pathways and promoted eNOS phosphorylation that was unaffected by Gβγ inhibition. These findings indicate that PI3-Kβ (regulated by Gβγ) may represent a novel molecular locus for eNOS activation by EDG receptors in vascular endothelial cells. These studies also indicate that different eNOS agonists activate distinct signaling pathways that diverge proximally following receptor activation but converge distally to activate eNOS.